Drosophila melanogaster Thor and response to Candida albicans infection

Abstract

We used Drosophila melanogaster macrophage-like Schneider 2 (S2) cells as a model to study cell-mediated innate immunity against infection by the opportunistic fungal pathogen Candida albicans. Transcriptional profiling of S2 cells coincubated with C. albicans cells revealed up-regulation of several genes. One of the most highly up-regulated genes during this interaction is the D. melanogaster translational regulator 4E-BP encoded by the Thor gene. Analysis of Drosophila 4E-BP(null) mutant survival upon infection with C. albicans showed that 4E-BP plays an important role in host defense, suggesting a role for translational control in the D. melanogaster response to C. albicans infection.

FIG. 1.

Interaction of Drosophila S2 cells with Candida strain CAI4-GFP. Drosophila S2 cells were incubated at 25°C with Candida at an MOI of 1 and monitored by time-lapse microscopy at ×400 magnification for the indicated times (bottom left, in hours). Arrows point to representative Candida cells engulfed by the S2 cells.

FIG. 2.

Phagocytosis of Candida strain CAI4-GFP by Drosophila S2 cells. Drosophila S2 cells were incubated at 25°C with Candida CAI4-GFP at an MOI of 1 for the indicated time. They were then stained with an anti-Candida polyclonal antibody (in red), as described in Materials and Methods. Engulfed Candida, protected from primary antibody binding, remained green, whereas nonphagocytosed Candida became yellow-red.

FIG. 3.

Northern blot analysis of Thor gene induction in S2 cells. Lanes from left to right: S2 cells (control), S2 cells infected with live wild-type C. albicans (SC5314), S2 cells in the presence of paraformaldehyde-fixed C. albicans, and S2 cells ingesting latex beads, for 3 and 6 h.

FIG. 4.

Western blot of d4E-BP protein in S2 cells. Lanes from left to right: S2 cells alone (control), S2 cells infected with live C. albicans (SC5314), S2 cells in the presence of paraformaldehyde-fixed C. albicans, and S2 cells ingesting latex beads for 6 h. Identical amounts of total protein (30 μg) were analyzed by Western blotting with 1868 antibody to d4E-BP or phospho-4E-BP1 (thr37/46). α, active, nonphosphorylated isoform; β, hyperphosphorylated isoform; actin, loading control.

FIG. 5.

Survival of D. melanogaster infected with C. albicans strain SC5314 is affected by the 4E-BP mutation. Survival of needle-pricked d4E-BP^null virgin flies was compared to the Oregon-R (wild type) and Thor^1rev1 (revertant) flies. (A) The d4E-BP^null male mutant flies were approximately two times more susceptible to the infection with C. albicans during the first 6 h than the wild-type and revertant flies. (B) The d4E-BP^null virgin female flies were 1.5 times more sensitive to the Candida infection than the wild-type and revertant female flies. As a control, d4E-BP^null, Oregon-R, and Thor^1rev1 flies of both genders were pricked with a sterile needle or with a needle coated with S. cerevisiae, which had no effect on the survival rate of Drosophila flies. Survival rates did not change significantly after 6 h. Each data point represents the mean of results from three independent experiments.